Project Summary Myasthenia gravis (MG) is an autoimmune neuromuscular disorder caused by autoantibodies that disrupt components of neuromuscular junctions, such as the acetylcholine receptor (AChR, 90% of patients). Rituximab (RTX) is a B cell depleting agent used in the management of an increasingly wide range of autoimmune diseases; many AChR MG patients achieve remission after RTX but relapse following treatment cessation is common. The thymus is a known reservoir of B cells that can produce pathogenic anti-AChR antibodies and resection of the thymus has long been known to improve MG symptoms; however, many patients also fail to achieve complete remission after thymectomy. In both cases, we conclude that the failed depletion of B cells relevant to disease may be driving poor responses. Developing systemic immunotherapy to better target B cells that escape depletion is therefore of critical importance for effective MG treatment. Characterizing the features of B cells that re-emerge after RTX depletion in MG would further the development of more effective treatments for MG and lead to a deeper understanding of the immuno-pathophysiology behind the disorder. The overall goal of this project is to characterize the transcriptional features of disease-relevant B cells from patients who experienced poor outcomes from two randomized clinical trials on the use of rituximab and thymectomy for the treatment of AChR MG. Our laboratory recently developed a method called single cell tracing of adaptive immune repertoires (STAIR) that allows for the unbiased identification and transcriptional characterization of autoantigen-specific B cells that escape RTX depletion by combining high-depth bulk repertoire sequencing methods with single cell gene expression and repertoire analysis. For this proposal, I will apply this approach to investigate the single cell gene expression characteristics of B cells from the thymus that persist in the circulation of patients who underwent thymectomy (Goal 1), and the characteristics of B cells associated with poor clinical responses to RTX (Goal 2). We will test the hypothesis that B cells shared with the thymus will have a similar single cell transcriptional identity as those that fail to be depleted by RTX: we expect that they will be clonally expanded IgG-switched ASCs with specificity for autoantigen. This fellowship integrates a training plan that will include invaluable learning experiences in machine learning and the latest NGS technology This training plan will enhance the applicant's goal of becoming a physician scientist at the interface of clinical medicine and science.